Immunological control of beta-amyloid levels in vivo

ABSTRACT

An antibody and vectorized antibody, capable of crossing the blood brain barrier, which catalyze hydrolysis of β-amyloid at a predetermined amide linkage are described. The antibody preferentially binds a transition state analog which mimics the transition state adopted by β-amyloid during hydrolysis. Also described are methods for sequestering free β-amyloid in an animal&#39;s bloodstream or for reducing β-amyloid or for disaggregating or preventing the formation of amyloid plaques in an animal&#39;s brain by administering β-amyloid specific antibodies or by immunization with endogenous β-amyloid epitopes. Methods which utilize or generate antibodies which catalyze the hydrolysis of β-amyloid for reducing levels of circulating β-amyloid in an animal; which generate antibodies that catalyze hydrolysis of a polypeptide by immunization with an epitope having a statine analog which mimics the polypeptide&#39;s hydrolysis transition state; and which utilize reduced peptide bond analogs to mimic the polypeptide&#39;s hydrolysis transition state are also provided.

BACKGROUND OF THE INVENTION

Alzheimer's disease is a progressive and ultimately fatal form ofdementia that affects a substantial portion of the elderly population.Definitive diagnosis at autopsy relies on the presence ofneuropathological brain lesions marked by a high density of senileplaques. These extracellular deposits are found in the neo-cortex,hippocampus and amygdala as well as in the walls of the meningeal andcerebral blood vessels. The principal component of these plaques is a 39to 43 residue β-amyloid peptide. Each plaque contains approximately 20fmole (80 picograms) of this 4 kDa peptide (Selkoe et al., J. ofNeurochemistry 46: 1820 (1986)). Apolipoprotein E and neurofibrillarytangles formed by the microtubule-associated tau protein are also oftenassociated with Alzheimer's disease.

β-amyloid is proteolytically cleaved from an integral membrane proteincalled the β-amyloid precursor protein. The gene which codes for thisprotein in humans is found on chromosome 21 (St George-Hyslop et al.,Science 235: 885 (1987), Kang et al., Nature 325: 733 (1987)). Numerouscultured cells and tissues (eg. brain, heart, spleen, kidney and muscle)express this β-amyloid precursor protein and also secrete the 4 kDaf-amyloid fragment into culture media, apparently as part of a normalprocessing pathway.

While it is difficult to establish an absolute causal relationshipbetween β-amyloid or the plaques it forms and Alzheimer's disease, thereis ample evidence to support the pathogenic role of β-amyloid. Forexample, patients with Down's syndrome have an extra copy of thef-amyloid precursor protein gene due to trisomy of chromosome 21 (StGeorge-Hyslop et al., Science 235: 885 (1987), Kang et al., Nature 325:733 (1987)). They correspondingly develop an early-onset Alzheimer'sdisease neuropathology at 30-40 years of age. Moreover, early-onsetfamilial Alzheimer's disease can result from mutations in the f-amyloidprecursor protein gene which fall within or adjacent to the f-amyloidsequence (Hardy, J., Nature Genetics 1: 233 (1992)). These observationsare consistent with the notion that deposition of β-amyloid as plaquesin the brain are accelerated by an elevation in its extracellularconcentration (Scheuner et al., Nature Med. 2: 864 (1996)). The findingthat β-amyloid is directly neurotoxic both in vitro and in vivo (Kowallet al., Proc. Natl. Acad. Sci. 88: 7247 (1991)), suggest that solubleaggregated β-amyloid, not the plaques per se, may produce the pathology.

Observations have indicated that amyloid plaque formation may proceed bya crystallization type mechanism (Jarrett et al., Cell 73: 1055 (1993)).According to this model, the seed that initiates plaque nucleation is anβ-amyloid which is 42 or 43 amino acids long (Aβ₁₋₄₃). Therate-determining nucleus formed by Aβ₁₋₄₃ or Aβ₁₋₄₂ allows peptidesAβ₁₋₄₀ or shorter to contribute to the rapid growth of an amyloiddeposit. This nucleation phenomenon was demonstrated in vitro by theability of Aβ₁₋₄₂ to cause the instantaneous aggregation of akinetically stable, supersaturated solution of Aβ₁₋₄₀. That finding hasled to the possibility that Aβ₁₋₄₀ might be relatively harmless in theabsence of the nucleation peptides Aβ₁₋₄₂ or Aβ₁₋₄₃. Indeed, elevatedlevels of these long peptides have been found in the blood of patientswith familial Alzheimer's disease (Scheuner et al., Nature Med. 2: 864(1996)). Moreover, Aβ₁₋₄₂ or Aβ₁₋₄₃ was found to be the predominant formdeposited in the brain plaques of many Alzheimer's disease patients(Gravina et al., J. of Biol. Chem. 270: 7013 (1995)).

Given the central role played by β-amyloid, it has become increasinglyimportant to understand the interrelationship between the differentpools of these molecules in the body. Free β-amyloid present in theblood most likely arises from peptide released by proteolytic cleavageof β-amyloid precursor protein present on cells in the peripheraltissues. Likewise most of the free β-amyloid found in the brain andcerebrospinal fluid is probably derived from peptide released bysecretase cleavage of β-amyloid precursor protein expressed on braincells. The peptides are identical regardless of origin, and the resultsfrom several studies suggest an intercommunication between these pools.

SUMMARY OF THE INVENTION

One aspect of the present invention is an antibody which catalyzeshydrolysis of β-amyloid at a predetermined amide linkage. In oneembodiment, the antibody preferentially binds a transition state analogwhich mimics the transition state adopted by β-amyloid during hydrolysisat a predetermined amide linkage and also binds to natural β-amyloidwith sufficient affinity to detect using an ELISA. In anotherembodiment, the antibody preferentially binds a transition state analogwhich mimics the transition state adopted by β-amyloid during-hydrolysisat a predetermined amide linkage, and does not bind natural f-amyloidwith sufficient affinity to detect using an ELISA. Antibodies generatedare characterized by the amide linkage which they hydrolyze. Specificantibodies include those which catalyze the hydrolysis at the amyloidlinkages between residues 39 and 40, 40 and 41, and 41 and 42, ofβ-amyloid.

Another aspect of the present invention is a vectorized antibody whichis characterized by the ability to cross the blood brain barrier and isalso characterized by the ability to catalyze the hydrolysis ofβ-amyloid at a predetermined amide linkage. In one embodiment, thevectorized antibody is a bispecific antibody. Preferably, the vectorizedantibody has a first specificity for the transferrin receptor and asecond specificity for a transition state adopted by β-amyloid duringhydrolysis. Specific vectorized antibodies include those which catalyzethe hydrolysis at the amyloid linkages between residues 39 and 40, 40and 41, and 41 and 42, of β-amyloid.

Another aspect of the present invention is a method for sequesteringfree β-amyloid in the bloodstream of an animal by intravenouslyadministering antibodies specific for β-amyloid to the animal in anamount sufficient to increase retention of β-amyloid in the circulation.Therapeutic applications of this method include treating patientsdiagnosed with, or at risk for Alzheimer's disease.

Another aspect of the present invention is a method for sequesteringfree β-amyloid in the bloodstream of an animal by immunizing an animalwith an antigen comprised of an epitope which is present on β-amyloidendogenous to the animal under conditions appropriate for the generationof antibodies which bind endogenous β-amyloid. Therapeutic applicationsof this method include treating patients diagnosed with, or at risk forAlzheimer's disease.

Another aspect of the present invention is a method for reducing levelsof β-amyloid in the brain of an animal by intravenously administeringantibodies specific for endogenous β-amyloid to the animal in an amountsufficient to increase retention of β-amyloid in the circulation of theanimal. In one embodiment, the antibodies are catalytic antibodies whichcatalyze hydrolysis of β-amyloid at a predetermined amide linkage. Theantibodies may be either monoclonal or polyclonal. In one embodiment,the antibodies specifically recognize epitopes on the C-terminus ofβ-amyloid₁₋₄₃.

Another aspect of the present invention is a method for reducing levelsof β-amyloid in the brain of an animal, by immunizing the animal with anantigen comprised of an epitope which is present on endogenous β-amyloidunder conditions appropriate for the generation of antibodies which bindendogenous β-amyloid. In one embodiment, the antigen is a transitionstate analog which mimics the transition state adopted by β-amyloidduring hydrolysis at a predetermined amide linkage. In a preferredembodiment, the antigen is comprised of Aβ₁₀₋₂₅. Preferably, theantibodies generated have a higher affinity for the transition stateanalog than for natural β-amyloid, and catalyze hydrolysis of endogenousβ-amyloid.

Similar methods which utilize or generate antibodies which catalyze thehydrolysis of β-amyloid for reducing levels of circulating β-amyloid inan animal, and also for preventing the formation of amyloid plaques inthe brain of an animal, are also provided. Also, methods fordisaggregating amyloid plaques present in the brain of an animal byutilizing or generating antibodies which catalyze the hydrolysis ofβ-amyloid are provided.

Another aspect of the present invention is a method for disaggregatingamyloid plaques present in the brain of an animal by intravenouslyadministering vectorized bispecific antibodies to the animal in anamount sufficient to cause significant reduction in β-amyloid levels inthe brain of the animal. The vectorized bispecific antibodies arecompetent to transcytose across the blood brain barrier, and have theability to catalyze hydrolysis of endogenous β-amyloid at apredetermined amide linkage upon binding. Preferably, the vectorizedbispecific antibodies specifically bind the transferrin receptor.

Another aspect of the present invention is a method for generatingantibodies which catalyze hydrolysis of a protein or polypeptide byimmunizing an animal with an antigen comprised of an epitope which has astatine analog which mimics the conformation of a predeterminedhydrolysis transition state of the polypeptide, under conditionsappropriate for the generation of antibodies to the hydrolysistransition state. This method can be used to generate catalyticantibodies to β-amyloid. A similar method, which utilizes reducedpeptide bond analogs to mimic the conformation of a hydrolysistransition state of a polypeptide, is also provided.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an amino acid sequence listing (SEQ ID NO: 1) of the 43residue β-amyloid peptide (Aβ).

FIG. 2 is an amino acid sequence listing (SEQ ID NO: 2) of the antigenicpeptide made from the N-terminal sequence of β-amyloid (Aβ₁₋₁₆).

FIG. 3 is an amino acid sequence listing (SEQ ID NO: 3) of the antigenicpeptide made from the central region of β-amyloid (Aβ₁₀₋₂₅). FIG. 4 isan amino acid sequence listing (SEQ ID NO: 4) (Aβ₃₅₋₄₃) of the antigenicpeptide made from the C-terminal sequence of β-amyloid.

FIG. 5 is a diagrammatic representation of data from an ELISA comparingmonoclonal antibody binding to Aβ₃₅₋₄₃ and Aβ₁₋₄₃ versus Aβ₁₋₄₀.

FIG. 6 indicates the amide linkages in the peptide made from theβ-amyloid C-terminal sequence (SEQ ID NO: 4) that were independentlyreplaced with a statyl moiety, to generate the different statinetransition state analogs of the peptide.

FIG. 7 indicates the amide linkages in the peptide made from theβ-amyloid central sequence (SEQ ID NO: 3) that were independentlyreplaced with a statyl moiety, to generate the different phenylalaninestatine transition state analogs of the peptide.

FIG. 8 is a structural comparison between the native β-amyloid peptideand the transition state phenylalanine statine β-amyloid peptide analog.

FIG. 9 is a structural comparison between the native β-amyloid peptideand the reduced peptide bond transition state β-amyloid peptide analog.

FIG. 10 is a formulaic representation of the native C-terminal region ofβ-amyloid, and the phosphonamidate transition state analog of theC-terminal region of β-amyloid (Aβ₃₅₋₄₃).

FIG. 11 indicates the putative transition state for peptide hydrolysisby zinc peptidases, compared to the phosphonate and phosphonamidatemimics.

FIG. 12 is a structural comparison of the native β-amyloid peptide andthe transition state phosphonamidate β-amyloid peptide which has thepeptide link between Gly 38 and Val 39 replaced with a phosphonamidatebond.

FIG. 13 is a diagrammatic representation of data from an ELISA whichassess the binding of monoclonal antibodies generated to transitionstate β-amyloid peptide analogs, to the normal Aβ₁₋₄₃ and to thephenylalanine statine transition state β-amyloid peptide.

FIG. 14 is a diagrammatic representation of data from an ELISA comparingantibody binding to the statine transition state β-amyloid peptideversus native β₁₋₄₃ and native Aβ₁₋₄₀. FIG. 15 is a graph of datashowing the cleavage of ¹²⁵I -Aβ-sepharose by monoclonal antibodiesgenerated to transition state analogs of β-amyloid.

FIG. 16 is a diagrammatic representation of data which quantitate theattachment of bispecific antibody to receptor-positive cells.

FIG. 17 is a diagrammatic representation of data obtained fromexperiments designed to track the transcytosis of vectorized bispecificantibody into brain.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to immunologically based methods forcontrolling levels of β-amyloid in the body of an animal. The inventionis based on the finding that antibodies specific for β-amyloid are ableto bind β-amyloid in the presence of a physiological level of humanserum albumin. The invention is also based on the finding that an animalcan tolerate the presence of antibodies specific for β-amyloid inamounts sufficient to sequester β-amyloid in the bloodstream.

One aspect of the present invention relates to a method for sequesteringfree β-amyloid in the bloodstream of an animal. The soluble andinsoluble forms of β-amyloid present within an animal are in dynamicequilibrium. Soluble β-amyloid is thought to translocate between bloodand cerebrospinal fluid. Insoluble β-amyloid aggregates deposit from thesoluble pool in the brain, as amyloid plaques. Results detailed in theExemplification section below indicate that intravenous administrationof antibodies specific for β-amyloid to an animal impedes the passage ofsoluble β-amyloid out of the peripheral circulation. This occurs becausethe β-amyloid specific antibodies, which are restricted to theperipheral circulation, bind to β-amyloid and sequester it in thecirculation. Such sequestration is accomplished through intravenousadministration of an appropriate amount of antibodies specific forβ-amyloid to the animal. The amount of antibody which is sufficient toproduce sequestration is dependent upon various factors (e.g., specificcharacteristics of the antibody to be delivered, the size, metabolism,and overall health of the animal) and are to be determined on a case bycase basis.

Administered antibodies can be monoclonal antibodies, a mixture ofdifferent monoclonal antibodies, polyclonal antibodies, or anycombination therein. In one embodiment, the antibodies bind to theC-terminal region of β-amyloid. Such antibodies specifically bind theless abundant, but more noxious Aβ₁₋₄₃ species in the blood as opposedto the smaller and less detrimental Aβ₁₋₄₀. In another embodiment, acombination of antibodies having specificity for various regions ofβ-amyloid are administered. In another embodiment, antibodies whichcatalyze the hydrolysis of β-amyloid, discussed in more detail below,are administered either alone or in combination with otheranti-β-amyloid antibodies.

The animal to which the antibodies are administered is any animal whichhas circulating soluble β-amyloid. In one embodiment, the animal is ahuman. The human may be a healthy individual, or alternatively, may besuffering from or at risk for a disease in which elevated β-amyloidlevels are thought to play a role, for example a neurodegenerativedisease such as Alzheimer's disease.

A related aspect of the present invention is a method for sequesteringfree β-amyloid in the bloodstream of an animal by stimulating an immuneresponse within the animal to endogenous β-amyloid. The results detailedin the Exemplification below indicate that an animal can tolerate theinduction of an immune response which produces antibodies to endogenousβ-amyloid, and that the presence of such antibodies will alter thedistribution of β-amyloid in the body, in a similar manner as the abovedescribed method of administering β-amyloid binding antibodies. Theimmune response to endogenous β-amyloid is generated by immunizing theanimal with one or more antigens comprised of epitopes present on theendogenous β-amyloid. Epitopes present on the inoculated antigens cancorrespond to epitopes present on any region of the β-amyloid molecule.In a preferred embodiment, epitopes found on the C-terminal region ofβ-amyloid are used to generate antibodies which specifically bind theAβ₁₋₄₃ species as opposed to the smaller Aβ₁₋₄₀. In an alternateembodiment, a combination of different epitopes are administered togenerate a variety of antibodies to β-amyloid. A more generalized immuneresponse is generated by immunizing either with a mixture of differentsmall peptide antigens or with the full-length 43 residue β-amyloidpeptide. In another embodiment, antigens used for inoculation includetransition state analogs of β-amyloid peptides to induce antibodieswhich have catalytic activity directed towards β-amyloid hydrolysis,described in detail below.

The immunoreactivity of the antigens can be enhanced by a variety ofmethods, many of which involve coupling the antigen to an immunogeniccarrier. In addition, various methods are known and available to one ofskill in the art for specifically enhancing the immunogenicity ofendogenous molecules or the epitopes contained therein. Variousmodifications can be made to the β-amyloid antigen(s) described hereinto render it more compatible for human use. For example, the peptide(s),can be genetically engineered into appropriate antigenic carriers, orDNA vaccines can be designed.

The above techniques for sequestering β-amyloid in the circulation arealso useful for reducing the levels of β-amyloid in the brain. Becausethe formation of amyloid plaques in the brain is dependent, at least inpart, on the levels of free β-amyloid present in the brain, reducingbrain β-amyloid levels of an animal will, in turn, reduce the formationof amyloid plaques in the brain. Therefore, the above techniques areuseful for preventing the formation of amyloid plaques in the brain ofan animal. This is especially applicable to an animal which isconsidered at risk for the development of amyloid plaques; a risk whichmay result from a genetic predisposition or from environmental factors.Administration of antibodies, or immunization of the animal to produceendogenous antibodies, to β-amyloid can be of therapeutic benefit tosuch an animal (e.g., a human who has a family history of Alzheimer'sdisease, or who is diagnosed with the disease).

Another aspect of the present invention relates to antibodies which arecharacterized by the ability to catalyze the hydrolysis of β-amyloid ata predetermined amide linkage. Experiments detailed in theExemplification section demonstrate the generation of differentantibodies which have proteolytic activity towards β-amyloid. Suchantibodies are generated by immunizing an animal with an antigen whichis a transition state analog of the β-amyloid peptide. A transitionstate analog mimics the transition state that β-amyloid adopts duringhydrolysis of a predetermined amide linkage. Transition state analogsuseful for generating the catalytic antibodies include, withoutlimitation, statine, phenylalanine statine, phosphonate,phosphonamidate, and reduced peptide bond transition state analogs.

Antibodies generated to epitopes unique to the transition statepreferentially bind β-amyloid in the transition state. Binding of theseantibodies stabilizes the transition state, which leads to hydrolysis ofthe corresponding amide bond. The particular amide linkage to behydrolyzed is chosen based upon the desired cleavage product. Forexample, cleavage of full length β-amyloid into two peptide fragmentswhich cannot aggregate into amyloid plaques would be of therapeutic usein the methods disclosed herein. Antibodies may be either monoclonal orpolyclonal. Several such transition state mimics have been made and usedas antigen in the generation of monoclonal antibodies which catalyze thecleavage at the indicated linkage. These antigens and the antibodiesgenerated are listed in Table 8 of the Exemplification section below.Antibodies generated to antigens which have transition state mimicsincorporated at a specific amide linkage, should bind the naturalhydrolysis transition states of these linkages in native β-amyloid,stabilizing the transition state and catalyzing cleavage at thatlinkage.

At least two different classes of antibodies are generated by the abovemethods. The first class preferentially binds the transition stateanalog, and also detectably cross reacts with natural β-amyloid usingthe ELISA detailed in the Exemplification section, to detect binding.The second class binds the transition state analog, and does notdetectably cross react with natural β-amyloid using the ELISA proceduredetailed in the Exemplification section to detect binding. Both classesof antibodies have potential value as catalytic antibodies. Therespective binding affinities of an anti-transition state antibody islikely to reflect its activity at catalyzing hydrolysis. It is thoughtthat in order for an antibody to have activity at catalyzing hydrolysisof a protein, it must possess at least a minimal ability to bind thenatural (non-transition) state of the protein. Antibodies which retainsignificant binding for β-amyloid, (that strongly cross react withnatural β-amyloid) may be more efficient at catalyzing hydrolysis due toa higher efficiency of binding the β-amyloid. Once bound, theseantibodies force the protein into a transition state conformation forhydrolytic cleavage. Alternatively, antibodies which only minimallycross react with natural β-amyloid, although less efficient at bindingnative β-amyloid, are likely to be more efficient at forcing the boundβ-amyloid into the transition state conformation for hydrolyticcleavage. It should be pointed out that failure to detect binding of theanti-transition state antibodies to natural β-amyloid by the ELISAmethods presented in the Exemplification herein does not necessarilyreflect an inability to bind natural β-amyloid sufficiently to functionas a catalytic antibody. More likely, a lack of detection merelyreflects the sensitivity limitations of the assay.

Antibodies which have substantial affinity for the predicted cleavageproducts of the native β-amyloid peptide may be subject to productinhibition and might therefore exhibit low turnover. Such undesirableantibodies can be identified by secondary screening using peptides whichcontain epitopes of the predicted cleavage products (e.g., via ELISA).

In a preferred embodiment, the antibodies are monoclonal. Monoclonalantibodies are produced by immunizing an animal (e.g., mouse, guineapig, or rat) with the transition state analog antigen, and subsequentlyproducing hybridomas from the animal, by standard procedures. Hybridomaswhich produce the desired monoclonal antibodies are identified byscreening. One example of a screening method is presented in theExemplification section which follows. In another embodiment, theantibodies are polyclonal. Polyclonal antibodies are generated byimmunizing an animal (e.g., a rabbit, chicken, or goat) with antigen andobtaining sera from the animal. Polyclonal antibodies which have thedesired binding specificities can be further purified from the sera byone of skill in the art through the course of routine experimentation.

Catalytic antibodies specific for β-amyloid can alternatively begenerated in an individual through the use of anti-idiotype vaccinesdesigned to elicit the production of catalytic antibodies. Such vaccinesare described in the disclosure of Raso and Paulus (U.S. patentapplication Ser. No. 09/102,451, ANTI-IDIOTYPE VACCINES TO ELICITCATALYTIC ANTIBODIES, filed by Applicants Jun. 22, 1998, currentlypending), the contents of which are incorporated herein by reference.

Another aspect of the present invention is the use of statine andreduced peptide bond analogs to elicit catalytic antibodies havingproteolytic activity. The Exemplification section below details methodsfor using statine analogs as antigen in the production of catalyticantibodies, and also lists examples of anti-transition-state antibodiesgenerated using these methods. The “statyl” moiety is derived fromnaturally evolved protease transition state inhibitors like amastatin,pepstatin, and bestatin. These naturally-occurring statine-basedinhibitors have been used to effectively block the activity ofaminopeptidases, aspartic proteases and the HIV protease. Syntheticpeptides containing a statine residue offer novel features for theinduction of catalytic antibodies. The statyl moiety has a tetrahedralbond geometry, its length is extended by two CH₂ units, it has astrategically placed OH group and the structure has no charge. Thepresence of the additional CH₂ units is expected to elicit a moreelongated antibody combining site, and antibodies possessing thisextended site will induce extra strain on the peptide substrate,producing an accelerated catalysis. In addition, the —OH group in thesestatine analogs is thought to better approximate the position andchemistry of the true transition state. Statine-based transition-stateanalogs should therefore elicit a class of antibodies which issignificantly different from those obtained from the more commonly usednegatively charged phosphonate analogs.

Reduced peptide bond analogs introduce a tetrahedral configuration,without increasing the distance between amino acid residues. Thisfeature should more closely approximate the true transition stategeometry, than previously used analogs. A positively charged secondaryamine replaces the amide nitrogen of the natural polypeptide and shouldelicit a complementary negatively charged side chain at a proximal locusin the antibody combining site. The presence of such ancillary glutamylor aspartyl groups on the antibody will assist antibody-mediatedcatalysis of peptide cleavage via acid-base exchange. Reduced peptidebond-based transition- state analogs should therefore elicit a class ofantibodies which is significantly different from those obtained fromusing the more commonly used negatively charged phosphonate analogs.Reduced peptide bond analogs and statine analogs can be used to producespecific transition state analog antigens for a wide variety of proteinsor polypeptides. These antigens can in turn be used to generate therespective catalytic antibodies.

Administration of the β-amyloid catalytic antibodies described above canbe used in the methods described above for 1) sequestering freeβ-amyloid in the bloodstream of an animal, 2) reducing levels ofβ-amyloid in the brain of an animal, and 3) preventing the formation ofamyloid plaques in the brain of an animal, to generate the analogousresults. Experiments presented in the Exemplification demonstrate thatimmunization of an animal with a transition state analog results in thegeneration of an immune response to produce antibodies which recognizethe transition state, and which catalyze hydrolysis of the β-amyloidprotein. This indicates that the transition state analogs can be used asantigens in these methods to induce the production of antibodies in theanimal which recognize and catalyze cleavage of endogenous β-amyloid.

Methods which involve reducing overall levels of β-amyloid in an animalthrough the proteolytic action of the above described catalyticantibodies are also encompassed by the present invention. The presenceof functional catalytic antibodies in the circulation of an animal willreduce the level of intact β-amyloid in the circulation by selectivehydrolytic cleavage. Accordingly, the present invention provides amethod for reducing levels of circulating β-amyloid in an animal byintroducing the above described catalytic antibodies into the animal.Administration of the antibodies to the animal is preferably viaintravenous administration. Such antibodies are either monoclonal, mixedmonoclonal, polyclonal or any mixture thereof. The origin of theantibody may affect the half-life of the antibody in the animal;antibodies from less related species are more likely to be recognized asforeign by the animal's immune system. Preferably, administeredantibodies are derived from a species closely related to the animal, tomaximize half-life and minimize adverse reactions by the host.Administration of isolated variable region antibody fragments mayproduce beneficial results in this regard.

The present invention also provides a method for reducing levels ofcirculating β-amyloid in an animal by immunizing the animal with aβ-amyloid transition state analog to induce endogenous catalyticantibody production. The use and design of such vaccines is describedabove, and detailed in the Exemplification section below.

The reduction of β-amyloid levels in the circulation of an animal isexpected to displace the equilibrium of β-amyloid in the body, andultimately lead to a reduction in the levels of β-amyloid in the brainof the animal through mass action. In this respect, the presentinvention provides methods for reducing the levels of β-amyloid in thebrain of an animal, by either administering catalytic antibodies to theanimal, or by administering a transition state analog to induceendogenous antibody production. It follows that these procedures alsohave value as methods for preventing the formation of amyloid plaques inthe brain of an animal, since the resulting reduction in the levels ofβ-amyloid in the brain of an animal should prevent the formation ofamyloid plaques. These procedures also have value as methods fordisaggregating amyloid plaques present in the brain of an animal, sinceevidence indicates that lower brain β-amyloid levels can lead to thedisaggregation of plaques.

Another aspect of the present invention provides a more direct method ofaltering the distribution of β-amyloid in the brain by actuallydelivering anti-β-amyloid antibodies to the brain. Methods describedabove for reducing levels of β-amyloid in the brain and for preventingaggregation of amyloid plaques depend upon exchange between β-amyloidpools in the blood, tissues, cerebrospinal fluid and the brain, theexchange being driven by an antibody-mediated disruption of theequilibrium between these different pools. In contrast, delivery ofanti-β-amyloid antibodies to the brain will directly affect β-amyloidaggregation. Evidence presented in the Exemplification section belowindicates that the binding of certain anti-β-amyloid antibodies inhibitsthe initial aggregation of β-amyloid in vitro, and also disaggregatespreformed in vitro β-amyloid complexes. Moreover, if insoluble peptideis in equilibrium with a low level of soluble β-amyloid, then ananti-β-amyloid binding antibody could upset this balance and graduallydissolve the precipitate. These observations indicate that the presenceof β-amyloid antibodies in the brain will directly inhibit the formationof amyloid plaques and will also disaggregate preformed plaques bydisrupting the dynamic equilibrium between soluble β-amyloid andfibrillar β-amyloid deposited as plaques. Furthermore, a highly activecatalytic antibody is expected to destroy insoluble β-amyloid plaques byhydrolytically cleaving the constituent aggregated peptides.

One way of delivering antibodies to the brain is by producing vectorizedantibodies competent for transcytosis across the blood-brain barrier.Vectorized antibodies are produced by covalently linking an antibody toan agent which promotes delivery from the circulation to a predetermineddestination in the body. Examples of vectorized molecules which cantraverse the blood-brain barrier are found in the prior art (Bickel etal., Proc. Natl. Acad. Sci. USA 90: 2618-2622 (1993); Broadwell et al.,Exp. Neurol. 142: 47-65 (1996)). In these examples, antibodies arelinked to another macromolecule, the antibodies being the agent whichpromotes delivery of the macromolecules. One example of such an agent isan antibody which is directed towards a cell surface component, such asa receptor, which is transported away from the cell surface. Examples ofantibodies which confer the ability to trancytose the blood-brainbarrier include, without limitation, anti-insulin receptor antibodies,and also anti-transferrin receptors (Saito et al., Proc. Natl. Acad.Sci. USA 92: 10227-31 (1995); Pardridge et al., Pharm. Res. 12: 807-816(1995); Broadwell et al., Exp. Neurol. 142: 47-65 (1996)). This firstantibody is covalently linked to an antibody which binds β-amyloid.Alternatively, coupling the β-amyloid antibodies to ligands which bindthese receptors (e.g., insulin, transferrin, or low density lipoprotein)will also produce a vectorized antibody competent for delivery to thebrain from the circulation (Descamps et al., Am. J. Physiol. 270:H1149-H1158 (1996); Duffy et al., Brain Res. 420: 32-38 (1987); Dehoucket al., J. Cell Biol. 138: 877-889 (1997)).

A vector moiety can be chemically attached to the anti-β-amyloidantibody to facilitate its delivery into the central nervous system.Alternatively, the moiety can be genetically engineered into theantibody as an integral component. This vector component can be forexample, an anti-transferrin receptor antibody or anti-insulin receptorantibody which binds the receptors present on the brain capillaryendothelial cells (Bickel et al., Proc. Natl. Acad. Sci. USA 90: 2618-22(1993); Pardridge et al., J. Pharmacol. Exp. Ther. 259: 66-70 (1991);Saito et al., Proc. Natl. Acad. Sci. USA 92: 10227-31(1995); Friden etal., J. Pharm. Exper. Ther. 278: 1491-1498 (1996)) which make up theblood-brain barrier. The resulting bifunctional antibody will attach tothe appropriate receptors on the luminal side of the vessel (Raso etal., J. Biol. Chem. 272: 27623-27628 (1997); Raso et al., J. Biol. Chem.272: 27618-27622 (1997); Raso, V. Anal. Biochem. 222: 297-304 (1994);Raso et al., Cancer Res. 41: 2073-2078 (1981); Raso et al., Monoclonalantibodies as cell targeted carriers of covalently and non-covalentlyattached toxins. In Receptor mediated targeting of drugs, vol. 82. G.Gregoriadis, G. Post, J. Senior and A. Trouet, editors. NATO AdvancedStudies Inst., New York. 119-138 (1984)). Once bound to the receptor,both components of the bispecific antibody pass across the blood-brainbarrier by the process of transcytosis. Anti-β-amyloid antibodies whichhave entered the brain interact directly with both β-amyloid plaques andthe soluble β-amyloid pool. It has been estimated that concentrations ofmacromolecules in the 10⁻⁸-10⁻⁷M range can be achieved in the brainusing vector-mediated delivery via these brain capillary enrichedprotein target sites (Maness et al., Life Sciences 55: 1643-1650 (1994);Lerner et al., Science 252: 659-667 (1991)). Importantly, the vectorappears safe since animals dosed daily for two weeks with ananti-transferrin receptor antibody displayed no loss of integrity of theblood-brain barrier, using a radioactive sucrose probe (Broadwell etal., Exp. Neurol. 142: 47-65 (1996)).

The Exemplification details the production of vectorized bispecificantibodies which bind β-amyloid. The bispecific antibodies transcytoseacross the blood brain barrier via a first specificity which binds thetransferrin receptor. Use of antibodies which bind the transferrinreceptor for delivery of agents across the blood brain barrier isdescribed by Friden et al. in U.S. Pat. No. 5,182,107; U.S. Pat. No.5,154,924; U.S. Pat. No. 5,833,988; and U.S. Pat. No. 5,527,527; thecontents of which are incorporated herein by reference.

Results from experiments presented in the Exemplification section whichfollows indicate that the produced bispecific antibodies retain theirseparate specificities and are delivered across the blood-brain barrierinto the brain parenchyma and brain capillaries of a live animal whenadministered intravenously.

Alternate methods for the production of bispecific antibodies have beendescribed for genetically engineering bispecific reagents or forproducing them intracellularly by fusing the two different hybridomaclones (Holliger et al., Proc. Natl. Acad. Sci. 90: 6444-6448 (1993);Milstein et al., Nature 305: 537 (1983); Mallander et al., J. Biol.Chem. 269: 199-206 (1994)). Vectorized bispecific antibodies produced bythese techniques can also be used in the methods of the presentinvention.

Since the introduction of whole antibodies into the brain might bedetrimental if they were to fix complement and promotecomplement-mediated lysis of neuronal cells, it may be beneficial toproduce and utilize smaller vectorized F(ab′)₂ bispecific reagents. Ithas been shown that aggregated β-amyloid itself can fix complement inthe absence of any antibody and that the resulting inflammation maycontribute to the pathology of Alzheimer's disease. The possibility ofintracerebral antibody having a similar effect can be greatly reduced byeliminating the Fc region of the antibody. Moreover, since coupling ofFab′ halves uses the intrinsic hinge region cysteines, no extraneoussubstituent linkage groups need be added. Faster or more efficient entryinto the brain represents another potential advantage that smallerF(ab′)₂or Fv₂ reagents may provide for intracerebral delivery. Inaddition, the two types of vectorized molecules may have differentbiodistribution and plasma half-life characteristics (Spiegelberg etal., J. Exp. Med. 121: 323 (1965)).

Depending on their design, anti-O-amyloid bispecific antibodies in thebrain can reduce soluble β-amyloid and β-amyloid deposits by threepotential mechanisms. An anti-β-amyloid bispecific antibody that tightlybinds soluble β-amyloid will not only sequester the peptide but, due toefflux of vectorized molecules from the central nervous system (Kang etal., J. Pharm. Exp. Ther. 269: 344-350 (1994)), may also carry the boundβ-amyloid out of the brain, releasing it into the blood stream. Such aclearance mechanism would lead to a continuous cycling of β-amyloid outof the brain. In addition, if the antibodies have catalytic activity,they will directly reduce the levels of harmful β-amyloid bydegradation. Since catalytic antibodies exhibit turnover, each antibodycan inactivate many β-amyloid molecules. Thus much less vectorizedbispecific antibody has to be delivered into the brain to achieve thedesired depletion of β-amyloid.

To be effective the anti-β-amyloid sites of a bispecific antibody mustbe empty before passage out of the blood and into the brain. Thereforethe concentration of bispecific antibody in animals must exceed thelevel of β-amyloid circulating in the blood. Calculations performedbased upon known β-amyloid levels (Scheuner et al., Nature Med. 2:864-870 (1996)) and a medium-range plasma level of bispecific antibodyexpected in a treated animal indicated 99.9% of the bispecificantibodies that enter the brain will have unoccupied anti-f-amyloidcombining sites.

Another way of delivering antibodies to the brain is via direct infusionof anti-β-amyloid antibodies into the brain of an animal. This techniquegives these antibodies immediate access to β-amyloid in the brainwithout having to cross the blood-brain barrier. Direct infusion can beaccomplished via direct parenchymal or intracerebroventricular infusion(Knopf et al., J. Immunol. 161: 692-701 (1998)). Briefly, the animal isanesthetized and placed in a stereotaxic frame. A midsagittal incisionis made on the scalp to expose the skull and the underlying fascia isscraped away. A hole is drilled to accept a sterilized length ofstainless steel hypodermic tubing, which is stereotaxically advanced sothat its tip is appropriately located in the brain. A guide cannula isthen attached to the skull and sealed. The cannula remains in place formultiple infusions of antibody into the brain. A bolus of a sterile 50mg/ml solution of a monoclonal anti-β-amyloid can be infused over a 2-8minute period into an immobilized animal via an injection cannula.

Delivery of catalytic antibodies into the brain of an animal via one ofthe above described methods, can also be used to disaggregate amyloidplaques present in the brain. The advantage of delivering anβ-amyloid-specific catalytic antibody into the brain is two-fold. Theβ-amyloid peptide is permanently destroyed by such antibodies and, sincecatalysis is continuous, each antibody inactivates many target β-amyloidmolecules in the brain. Thus much less antibody has to be infused intothe central nervous system to achieve the desired depletion ofβ-amyloid.

The amount of antibody to be administered or delivered to the animalshould be sufficient to cause a significant reduction in β-amyloidlevels in the brain of the animal. The appropriate amount will dependupon various parameters (e.g., the particular antibody used, the sizeand metabolism of the animal, and the levels of endogenous β-amyloid)and is to be determined on a case by case basis. Such determination iswithin the means of one of average skill in the art through no more thanroutine experimentation.

It is expected that additional benefits with respect to lowering brainβ-amyloid levels and preventing or disaggregating amyloid plaques can beachieved through utilizing a combination of one or more of the abovedescribed approaches.

Exemplification

Section 1: Retention of β-Amyloid in the Circulation

Synthesis of β-Amyloid Peptide Antigens

The amino acid sequence of the 43 residue β-amyloid peptide (A,8)islisted in FIG. 1. To determine which sites on this A:i peptide were bestsuited for antibody-mediated therapy, three key regions (amino-terminal,central and carboxy-terminal) of the A: 43-mer were chosen to generateepitope-specific vaccines. These shortened peptides served as antigenicepitopes to induce a highly specific antibody response.

Monoclonal antibodies to the amino-terminal region of A have been shownin the past to have the ability to solubilize Aβ aggregates (Solomon etal., Proc. Natl. Acad. Sci. USA 94(8): 4109 (1997); Solomon et al.,Proc. Natl. Acad. Sci. USA 93(1): 452 (1996)). A peptide consisting ofthe amino-terminal region of A: was similarly designed for the presentexperiments (shown in FIG. 2 and listed in SEQ ID NO: 2) and used toelicit amino-terminal specific antibodies that bind Aβ. A Cys residuewas added to the C-terminus of the Aβ sequence to provide a suitablelinkage group for coupling this peptide to an antigenic carrier proteinsuch as maleimide-activated Keyhole Limpet Hemocyanin (KLH).

A peptide encompassing the central region of Aβ was also synthesized(shown in FIG. 3 and listed in SEQ ID NO: 3). A Cys residue was placedat the N-terminus of the Aβ sequence to provide a sulfhydryl linkagegroup for coupling the peptide to antigenic carrier proteins such asmaleimide-activated KLH.

To produce an antigen for eliciting an immune response directed againstthe carboxy-terminus of Aβ (Suzuki et al., Science 264: 1336 (1994)), adecapeptide encompassing the N-terminal region of Aβ, with an additionalCys residue at the N-terminus, was synthesized (Shown in FIG. 4, andlisted in SEQ ID NO: 4). The Cys substitution was designed to provide asulfhydryl linkage group for coupling the peptide to antigenic carrierproteins such as KLH.

Coupling the Peptides to an Antigenic Carrier Protein

The different Cys containing Aβ peptides were individuallythioether-linked to maleimide-activated KLH. A multivalent Aβ vaccinewas also produced by simultaneously linking all three of these peptidesto maleimide-activated KLH. In addition the full-length Aβ 43-mer waslinked to KLH using glutaraldehyde.

Antibodies Elicited with the β-Amyloid Vaccines

Normal BALB/c mice were immunized by standard procedures with theKLH-linked Aβ vaccines described above. The mice were either bled orsacrificed for removal of the spleen for hybridoma production. Sera andmonoclonal antibodies obtained were characterized for binding to Aβ.

Table 1 shows the results from an ELISA run with 1/100 diluted serumfrom two non-immunized control mice versus 1/100 and 1/1000 dilutedserum from a mouse that was immunized with a central region ASpeptide-KLH vaccine. The free Aβ peptide was adsorbed directly onto themicrotitre plate to avoid detection of anti-KLH antibodies in the serum.TABLE 1 ELISA for Binding to the Central Region Aβ Peptide AntibodyBound Addition (O.D. 450 nm) Control Serum A 1/100 0.666 Control Serum B1/100 0.527 Mouse 1 antiserum 1/100 3.465 Mouse 1 antiserum 1/1000 2.764

Monoclonal antibodies raised against this central region Aβ peptide andproduced by hybridoma fusion were identified using the above describedELISA assay. A binding assay was performed to determine whether themonoclonal anti-Aβ antibodies identified also bound to the full length Mpeptides. ¹²⁵I-Aβ₁₋₄₃ probe was incubated with hybridoma secretions fromthe indicated clones. A standard polyethylene glycol separation methodwas used to detect ¹²⁵I-Aβ₁₋₄₃ bound antibody (Table 2). Resultspresented in Table 2 indicate that the antibodies generated to thepeptide fragments also bound full length Aβ₁₋₄₃. TABLE 2 ¹²⁵I-Aβ₁₋₄₃Binding Assay ¹²⁵I-Aβ₁₋₄₃ Bound Addition (cpm) Control Hy 3,171 ControlHy 2,903 6E2 15,938 6E2 1/10 9,379 3B1 12,078 3B1 1/10 3,353 8E3 10,7898E3 1/10 3,249

It was reported that when ¹²⁵I-Aβ₁₋₄₀ is added to human plasma, ˜89%binds to albumin (Biere et al., J. of Biol. Chem. 271(51): 32916(1996)). This raises the concern that the bound albumin will interferewith antibody binding. Binding assays were performed in the presence andabsence of serum albumin, to determine whether albumin bindinginterferes with antibody binding to Aβ. The ability of purified 5A11monoclonal anti-Aβ antibody to bind ¹²⁵I Aβ₁₋₄₀ was unaffected by thepresence of human serum albumin (HSA) 10 at 60 mg/ml, even though thiswas a 500-fold molar excess over the antibody concentration (Table 3).These results indicate that the ability of antibodies to bind to andsequester Aβ in the blood will not be attenuated by the presence ofother binding proteins. TABLE 3 ¹²⁵I-Aβ₁₋₄₀ Binding to Antibody in thePresence of Human Serum Albumin* ¹²⁵I-Aβ₁₋₄₀ Bound Specifically BoundAddition (cpm) (% of total added) Control 8,560 — +5A11 anti-Aβ 64,58979 Control + HSA* 3,102 — +5A11 anti-Aβ + HSA* 55,304 75*HSA at 60 mg/ml (˜1 mM); anti-Aβ 5A11 at 2 × 10⁻⁶ M; Added ˜70,000 cpmof ¹²⁵I-Aβ₁₋₄₀Monoclonal Antibody Production

A mouse was immunized with a KLH conjugate of the central region Aβ₁₀₋₂₅peptide (This peptide antigen had a phenylalanine statine transitionstate analog at an amide linkage, discussed further in Section II,below). A hybridoma fusion was performed and the resulting monoclonalantibodies analyzed to characterize the specificity of the immuneresponse to the vaccine. Hybridoma supernatants produced in the fusionwere screened using ELISA to assess their binding to the Aβ₁₋₄₃ peptide.

The monoclonal antibodies produced were determined to bind to the Aβ₁₋₄₃peptide adsorbed directly onto an ELISA plate. Strong color reactionswere obtained in this ELISA using only 10 μl of hybridoma supernatantwhile the addition of media alone produced low background color. Theseresults indicate that the antibodies not only bound to the small peptideimmunogen but they were also reactive with the full-length Aβ₁₋₄₃.Importantly, antibodies bound to the carrier-free Aβ peptide adsorbeddirectly onto microtitre plates, showing their specificity for thepeptide rather than the immunogenic carrier. The high affinity 5A11monoclonal antibody (Table 3) was obtained from this hybridoma fusion.

A second mouse was immunized with a KLH conjugate of the Aβ₃₅₋₄₃ analogencompassing the C-terminal region of Aβ. Serum from the mouse wasscreened for reaction with Aβ₁₋₄₃ adsorbed directly onto the ELISAwells. The assay results are presented in Table 4. The spleen of thismouse was then used for a hybridoma fusion to further characterize thespecificity of its immune response. Importantly, none of the miceimmunized with Aβ vaccines or the anti-Aβ ascites-producing micedisplayed ill effects even though some of those induced antibodiescross-react with mouse Aβ and mouse amyloid precursor protein. TABLE 4ELISA for Binding of Antiserum Directed to the Carboxy-terminal AβPeptide Antibody Bound (O.D. 450 nm) Addition Native Aβ₁₋₄₃ ControlSerum 0.484 Mouse Antiserum 1.765

Monoclonal antibodies from hybridoma clones generated above werescreened for binding to the small carboxy-terminal peptide Aβ₃₅₋₄₃ andthe full-length Aβ₁₋₄₃. Results are presented in FIG. 5. The monoclonalantibodies bound to the carboxy-terminal locus on each of thesecarrier-free Aβ peptides adsorbed directly to the microtitre plate,confirming their specificity for the peptide rather than the immunogeniccarrier. The clones were also tested with Aβ₁₋₄₀ to identify antibodieswhich do not react with this shortened, 40 amino acid residue version ofAβ and thus will specifically bind to the carboxy-terminus of Aβ₁₋₄₃(FIG. 5). Used therapeutically, this vaccine should elicit antibodieswhich will preferentially bind the less abundant, but more noxiousAβ₁₋₄₃ species in the blood as opposed to the smaller and lessdetrimental Aβ₁₋₄₀.

In a separate experiment, mice were immunized with a vaccine comprisedof a cocktail of the three distinct KLH-peptide antigens (FIGS. 2-4)representing the distinct regions of β-amyloid (FIG. 1). Control micewere immunized with KLH alone. The antigens were emulsified in completeFreunds adjuvant prior to the first injection and in incomplete Freundsadjuvant for subsequent injections. Tests were performed on dilutedserum from these Aβ-KLH immunized mice to determine the presence ofspecific anti-Aβ antibodies. The Aβ₁₋₁₆, Aβ₁₄₋₂₅, Aβ₃₄₋₄₃, Aβ₁₋₄₀, andAβ₁₋₄₃ peptides were used to identify antibody specificity. The peptideswere adsorbed directly onto an ELISA plate. The results are presented inTable 5. The results indicate that mice immunized with the cocktail ofthe three peptide antigens produced serum containing antibodies whichreact with the amino-terminal, central region, and carboxyl-terminalpeptides, as well as with the full-length Aβ 40-mer and 43-mer. Theconstant presence of this spectrum of anti-Aβ antibodies will be veryeffective in binding all of the soluble Aβ in the peripheral circulationof a vaccinated animal. TABLE 5 ELISA to Measure the Serum AntibodiesPresent in Immunized Mice ELISA READING (O.D. 450 nm) Immunogen Aβ₁₋₁₆Aβ₁₄₋₂₅ Aβ₃₄₋₄₃ Aβ₁₋₄₀ Aβ₁₋₄₃ Mouse 1 (Control) KLH 0.076 0.038 0.0640.042 0.066 Mouse 2 Aβ-KLH 3.013 1.258 3.191 2.337 2.598 Cocktail Mouse3 Aβ-KLH 1.484 1.180 2.068 1.758 1.680 Cocktail Mouse 4 Aβ-KLH 1.4861.072 2.276 1.444 1.709 CocktailVaccine Trials in Non-Human Primates

Given the potential importance of β-amyloid vaccine therapy for humanpatients of Alzheimer's disease, a human-compatible, alum-based Aβpeptide vaccine preparation has been tested in non-human primates.Antibody production and safety studies for the human-compatibleβ-amyloid vaccines have commenced in Cynomolgus monkeys (Macacafascicularis). This animal system is highly relevant to humanapplications since the predicted amino acid sequence of β-amyloid inthese primates is identical to humans, and their basic physiology andimmunological systems closely approximate those which will beencountered in a clinical situation. Cynomolgus monkeys were vaccinatedmonthly and were periodically bled to monitor anti-Aβ levels in theserum. The monkeys were also observed for any ill effects.

The Cynomolgus monkeys mounted a strong immune response to a singleinjection of the simplest vaccine preparation composed of the fulllength β-amyloid peptide adsorbed to an aluminum hydroxide gel. Thespecificity of those early anti-β-amyloid antibodies was characterizedby ELISA using various Aβ peptide fragments (Table 6). This analysisindicated that the monkeys produced antibodies that bind to thefull-length peptide and react with its amino-terminal, central andcarboxyl-terminal regions. TABLE 6 ELISA to Measure the Serum AntibodiesPresent in Aβ Vaccinated Macaca fascicularis ELISA READING (O.D. 450 nm)Vaccination Schedule Aβ₁₋₁₆ Aβ₁₄₋₂₅ Aβ₃₄₋₄₃ Aβ₁₋₄₀ Aβ₁₋₄₃Pre-Vaccination 0.511 0.404 0.370 0.380 0.235 Aβ/Alum (1st month) 2.1151.687 0.671 2.393 2.479

Importantly, the vaccinated monkeys are perfectly healthy and appearcompatible with the anti-M antibodies that have been circulating intheir body for over three months. Thus far, there are no apparent sideeffects due to cross-reaction of the anti-Aβ antibodies with naturallyoccurring β-amyloid precursor protein or other vital components. Theseanimals were closely observed by a veterinarian, and have exhibited nosigns of autoimmune disease, immune complex disease or any otheradverse/toxic reaction to the vaccination.

In continuing experiments boost injections will be performed as perusual methods. The sera produced will be monitored for antibodyspecificity and affinity parameters as the immune response intensifiesand matures. At termination, a complete necropsy and histopathologicalexamination will be performed on the monkeys. Genetically engineered Aβvaccines, discussed below, will also be evaluated in the Cynomolgusmonkeys to determine if they will prove to be even better immunogens.

Antibodies Affect the Distribution of ¹²⁵I-Aβ in Normal Mice

Anti-Aβ antibodies in the circulation cannot cross the blood-brainbarrier to a significant extent and therefore should act as a sink thatprevents ¹²⁵I-β₁₋₄₀ from reaching the brain. This retention effect wasdemonstrated by measuring the blood levels in mice 4 h after injectingthem with equal amounts of ¹²⁵I-Aβ₁₋₄₀either alone or along with our5A11 anti-Aβ monoclonal antibody (Table 7). The passage of ¹²⁵I-Aβ₁₋₄₀out of the peripheral circulation was greatly curtailed in animals whichconcomitantly received the specific anti-Aβ antibody. That findingextends the in vitro results obtained with the 5A11 antibody (Table 3)by demonstrating the antibody can effectively bind Aβ in an experimentalanimal. The observation that animals treated with this antibody retained10-times more ¹²⁵I-Aβ₁₋₄₀ in the circulation indicates that theequilibrium distribution of Aβ in the body can be dramatically alteredby selective sequestration in the blood. TABLE 7 Anti-Aβ AntibodyImpedes the Passage of ¹²⁵I-Aβ₁₋₄₀ Out of the Circulation ¹²⁵I-Aβ₁₋₄₀ inBlood Mouse Injected With; (cpm/gm) ¹²⁵I-Aβ₁₋₄₀ alone 27,300¹²⁵I-Aβ₁₋₄₀ + 5A11 anti-Aβ 278,900Genetically Engineered Vaccines

Genetically engineered β-amyloid antigen vaccines for use in humans arecurrently being developed in order to induce protective levels ofanti-β-amyloid antibodies. β-amyloid fragments will be engineered intochimeric Aβ vaccines which incorporate highly immunogenic carriermoieties to increase the appropriate antigenic response in a humanpatient. Carrier moieties suitable for use include diphtheria toxoid(DT) and the hepatitis B core antigen (HBcAg). These represent powerfuldelivery systems for β-amyloid peptides, and are known to induce anexcellent, high titer immune response when used with alum as anadjuvant.

DT is licensed for use as a conjugate vaccine for H. influenzae type Band renders this immunogen T-cell dependent. The expression of DT inrecombinant E. coli is high. One or more of the above describedβ-amyloid peptides will be fused at the C-terminus of the catalyticdomain of DT, or at either end of the combined transmembrane/receptorbinding domains of DT. The produced fusions will be used with analuminum hydroxide gel adjuvant to generate potent vaccines.

High titers of antibody directed against heterologous epitopes have beenproduced using the HBcAg delivery systems and aluminum hydroxide geladjuvant. HBcAg has several distinct advantages as a fusion partner forAβ peptides. The immunodominant internal site between amino acids 75 and81 can accommodate heterologous sequences up to 45 amino acids. The coreself-assembles into larger 27 nm particles that are highly immunogenic.Furthermore, HBcAg can be produced in recombinant E. coli at elevatedlevels.

The genetically engineered vaccines produced will be tested foreffectiveness in depleting or preventing plaques using mouse and otherrelevant animal models. Antibody production and safety trials for thevaccines will be conducted in Cynomolgus monkeys.

Methods of the Invention

Peptide synthesis. The 40mer Aβ₁₋₄₀, the 43mer Aβ₁₋₄₃, and the threesmall Aβ peptides Aβ₁₋₁₆, Aβ₁₀₋₂₅, and Aβ₃₅₋₄₃, were synthesized bystandard automated Fmoc chemistry. Newly synthesized peptides werepurified by HPLC and their composition was verified by mass spectral andamino acid analysis. The Aβ 43mer was obtained from a commercial source(Bachem, Torrance, Calif.).

Conjugation of β-Amyloid Peptides to Immunogenic Carriers.

The small Aβ peptides were linked to the KLH carrier protein in order torender them antigenic. A Cys residue was strategically placed at the N—or C-terminal end of these A peptides to provide a suitable linkagegroup for coupling them via a thioether bond to maleimide activatedcarrier proteins. This linkage is stable and attaches the peptide in adefined orientation. Addition of ˜20 peptides/KLH is typically obtainedby this conjugation method. The longer, full length Aβ peptides werelinked to carrier proteins using a glutaraldehyde coupling procedure.

β-amyloid Antigen Cocktail. The three Aβ peptides shown in FIGS. 2-4were each individually conjugated to KLH. 20 μg of each of these threeconjugates was then mixed together. This mixture was emulsified withcomplete Freunds adjuvant and injected i.p. into mice. Subsequentmonthly i.p. booster injections used the same cocktail mixtureemulsified in incomplete Freunds adjuvant. Control mice received asimilar immunization protocol but using KLH which had not beenconjugated with the Aβ peptides.

Immunization of Mice. Normal BALB/c mice were immunized by standardprocedures with the KLH-linked A: vaccines described above. Briefly,mice were injected i.p. with antigen emulsified in complete Freundsadjuvant, followed by a second course in incomplete Freunds adjuvant.The mice were i.v. boosted with antigen in PBS three days prior tobleeding them or removing the spleen for hybridoma fusions to producemonoclonal antibodies.

None of the mice immunized with AS vaccines or the anti-Aβascites-producing mice displayed ill effects even though some of theantibodies cross-reacted with mouse and mouse amyloid precursor protein.

ELISA. The presence of bound anti-peptide antibodies was revealed byusing a peroxidase-labeled anti-mouse IgG probe followed by thechromogenic substrate (Engvall et al., Immunochemistry 8: 871-875(1971)).

Binding Assay. Both Aβ₁₋₄₃ and Aβ₁₋₄₀ were radiolabeled with ¹²⁵I. Theiodinated peptide was separated from unlabeled material by HPLC to giveessentially quantitative specific activity (˜2000 Ci/mmol) (Maggio etal., Proc. Natl. Acad. Sci. 89: 5462 (1992)). ¹²⁵I-Aβ₁₋₄₃probe wasincubated for 1 h at 23° C. with Hy media taken from hybridoma clonesproducing monoclonal anti-Aβ antibodies. A standard polyethylene glycolseparation method was used to detect the amount of ¹²⁵I-Aβ₁₋₄₃ bound toantibody.

β-Amyloid Vaccines for Primates. The immunogen used was a sytheticAβpeptide encompassing amino acids 1-41 of the Aβ protein. This peptidewas purified by HPLC and freeze-dryed and then resuspended in sterilewater at a concentration of 1.5 mg/ml. The vaccine was prepared bymixing 7.5 ml of a 2% aluminum hydroxide gel adjuvant (Alhydrogel,Superfos Biosector, Denmark), referred to herein as alum gel, with 7.5ml of the peptide. Tests showed that all of the peptide was adsorbed tothe alum gel after mixing for 12 hours at 25° C.

Monkeys were initially vaccinated by intramuscular (i.m.) injection of0.5 ml of the alum-adsorbed peptide. A second vaccination (boost) of thesame vaccine preparation (0.5 ml) was administered a month later.Subsequent identical monthly injections (boosts) will be given until theexperiment is terminated.

Genetically Engineered Vaccines. Highly immunogenic carrier moietieswill be used to construct chimeric Aβ vaccines. Moieties used willinclude diphtheria toxoid (DT) and the hepatitis B core antigen (HBcAg).The HBcAg expression system will be utilized (Schodel et al., Infect.and Immun. 57: 1347-1350 (1989); Schodel et al., J. of Exper. Med. 180:1037-1046 (1994); Schodel et al., J. of Virology 66: 106-114 (1992);Milich et al., Annals New York Academy of Sciences: 187-201 (1993)). Theamino terminal end of the catalytic domain of HBcAg has a signalsequence which should allow the Aβ fusion protein to be secreted intothe culture medium. The culture medium will be concentrated using alarge Amicon ultrafiltration device, and the concentrate thenchromatographed on a large Superdex 75 column. Recombinant productsobtained from within lysed cells will be separated from bacterialprotein using a combination of anion exchange and size exclusion FPLC.

Section II: Eliciting Monoclonal Antibodies With Transtion StateAntigens

Transition State Peptide Antigens

Different types of transition state peptide antigens were synthesized touse in the generation of antibodies which preferentially recognize(hydrolysis) transition states of Aβ at a predetermined amide linkageposition.

A series of statine (Sta) transition state analogs encompassing thecarboxy-terminal region of Aβ(Cys-Met-Val-Gly-Gly-Val/Sta-Val/Sta-Ile/Sta-Ala-Thr) were synthesized.Replacement of the proposed scissile peptide linkage between Val₃₉ andVal₄₀, Val₄₀ and Ile₄₁, and Ile₄₁ and Ala₄₂, with a “statyl” moiety(—CHOH—CH₂—CO—NH—) was designed to elicit catalytic antibodies thathydrolytically cleave Aβ at one of these sites (FIG. 6). A Cys residuewas placed at the N-terminal position of these peptides to provide asuitable linkage group for coupling to a maleimide-activated carrierprotein.

A series of phenylalanine statine (PhSta) transition state analogsencompassing the central region of Aβ(Cys-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe/PhSta-Phe/PhSta-Ala-Glu-Asp-Val-Gly-amide)was synthesized in this laboratory.

Replacement of the proposed scissile peptide linkage between Phe₁₉ andPhe₂₀, and between Phe₂₀ and Ala₂₁, with a statyl moiety(—CHOH—CH₂—CO—NH—) was designed to elicit catalytic antibodies thathydrolytically cleave M at these sites (FIG. 7). A Cys residue wasplaced at the C-terminus of these peptides to provide a sulfhydryllinkage group for coupling the peptides to antigenic,maleimide-activated carrier proteins such as KLH.

A structural comparison (FIG. 8) was made between the native Aβ peptideand the transition state phenylalanine statine Aβ peptide using agraphics workstation. An energy minimization algorithm (2000 iterations)was applied to arrange each peptide in its most favorable conformation.

The peptide link (—CO—NH—) between Phe₁₉ and Phe₂₀ was replaced with anelongated “statyl” moiety (—CHOH—CH₂—CO—NH—) and an energy minimizationwas applied. This orientation shows the difference between the planarpeptide link (—CO—NH—) of natural Aβ (left) versus the extended,tetrahedral “statyl” moiety (—CHOH—CH₂—CO—NH—) in the transition statepeptide (right).

An antibody combining site complementary to a tetrahedral statinetransition state analog will force the planar peptide bond of the Aβsubstrate into a transition state-like conformation. Such distortionshould catalyze the cleavage of Aβ at that locus in the peptidesequence.

The possibility of using a reduced peptide bond linkage to mimic thetransition state during hydrolysis of an amide linkage was alsoexplored. A reduced peptide bond linkage can be easily placed at almostany site in the Aβ molecule to produce a reduced peptide bond transitionstate analog. This analog can also be used to elicit catalyticantibodies that will hydrolytically cleave Aβ at the chosen site. Thereduced peptide bond transition state Aβ analog made was the(Gln-Lys-Leu-Val-Phe-CH₂—NH₂ ⁺-Phe-Ala-Glu-Asp-Val-Gly-Cys-amide)central region peptide; [calculated 1,342 (M+1); observed 1,344].

A structural comparison (FIG. 9) was made between the native Aβ peptideand the reduced peptide bond transition state Aβ analog using a graphicsworkstation. The peptide link (—CO—NH—) between Phe₁₉ and Phe₂₀ wasreplaced with a reduced peptide bond (—CH₂—NH₂ ⁺—) and an energyminimization was applied. The orientation shown indicates the differencebetween the planar peptide link (—CO—NH—) of natural Aβ (left) versusthe corresponding tetrahedral moiety (—CH₂—NH₂ ⁺—) in the reducedpeptide bond transition state analog (right). An energy minimizationalgorithm (2000 iterations) was applied to arrange each peptide in itsmost favorable conformation.

A phosphonamidate transition state analog of the carboxy-terminal regionof Aβ has also been synthesized (FIG. 10). Replacement of the proposedscissile peptide linkage between Gly₃₈ and Val₃₉ with a phosphonamidatemoiety (—PO₂ ⁻—NH—) was designed to elicit catalytic antibodies thatwill hydrolytically cleave A at this site. The N-acetyl-Cys residue wasplaced at the position of Leu₃₄ to provide a suitable linkage group forcoupling this peptide to an antigenic carrier protein. The structures inFIG. 11 represent the putative transition state for peptide hydrolysisby zinc peptidases, versus structure of and the phosphonate andphosphonamidate mimics. Similar tetrahedral transition stateintermediates are known to be formed by reaction with each of the fourclasses of proteolytic enzymes, the serine-, cysteine-, aspartic- andmetallo-peptidases.

A structural comparison was made between the native To peptide and thetransition state phosphonamidate Aβ peptide (FIG. 12) using a graphicsworkstation. The peptide link (—CO—NH—) between Gly₃₈ and Val₃₉wasreplaced with a phosphonamidate bond (—PO₂ ⁻—NH—) and an energyminimization was applied. The orientation shown in FIG. 12 illustratesthe difference between the planar peptide link (—CO—NH—) of native Aβ(left) versus the corresponding tetrahedral phosphonamidate bond (—PO₂⁻—NH—) in the transition state peptide (right).

An antibody combining site complementary to the tetrahedral transitionstate analog on the right of FIG. 12, will force the normally planarbond of the Aβ substrate peptide on the left into a transitionstate-like conformation. Such bond distortion is expected to catalyzethe hydrolytic cleavage of the Aβ peptide at the Gly₃₈-Val₃₉ linkage.

Immunization with Transition State Peptide Antigens

Peptide antigens were coupled to the immunogenic carrier KLH prior toimmunization of mice. Standard protocols were used to immunize BALB/cmice with the KLH-linked Aβ peptides described in the precedingsections. Briefly this procedure used i.p. injection of the differentantigens emulsified in complete Freunds adjuvant, followed by a secondcourse in incomplete Freunds adjuvant. Three days prior to hybridomafusion, the BALB/c mice were boosted i.v. with antigen in PBS.

A hybridoma fusion was performed using the spleen of a mouse immunizedwith either a mixture of the phenylalanine statine transition stateantigens generated (FIG. 7), a mixture of the statine (Sta) transitionstate Aβ antigens generated (FIG. 6), the reduced peptide bondtransition state Aβ antigen generated (transition state mimic locatedbetween Phe₁₉-Phe₂₀), or the phosphoamidate transition state Aβ antigengenerated (transition state mimic located between Gly₃₈-Val₃₉).Monoclonal antibodies listed in Table 8 were generated from these mice.TABLE 8 Potential Bonds Cleavage Antibodies Analog Used Modified SitesGenerated statine Val₃₉-Val₄₀ Val₃₉-Val₄₀ 2B2, 2H6, 3F2, Val₄₀-Ile₄₁Val₄₀-Ile₄₁ 4D3, 6A6, 1E4, Ile₄₁-Ala₄₂ Ile₄₁-Ala₄₂ 11E9, 9D6, 5C7, 7C7,1D12 phenylalanine- Phe₁₉-Phe₂₀ Phe₁₉-Phe₂₀ 6E2, 5A11, 6F11, statinePhe₂₀-Ala₂₁ Phe₂₀-Ala₂₁ 2E3, 8E3, 5G4, 4C7, 8D12, 2C12, 4G7, 5C7, 3C1,4H9, 8E6, 1H2, 3B1, 2H11 reduced Phe₁₉-Phe₂₀ Phe₁₉-Phe₂₀ 6E7, 6F6peptide bond phosphoamidate Glu₃₈-Val₃₉ Gly₃₈-Val₃₉ in progressDemonstration of Aβ Binding by Generated Antibodies

It was very important to demonstrate that the anti-Aβ andanti-transition state Aβ monoclonal antibodies bound to the naturalAβ₁₋₄₃ peptide which they were designed to sequester or cleave. To dothis, Aβ₁₋₄₀ and Aβ₁₋₄₃were radiolabeled with ¹²⁵I and the iodinatedpeptide was then separated from unlabeled material by HPLC. Probe wasincubated with either purified anti-Aβ antibodies or media taken fromhybridoma clones producing anti-Aβ antibodies. The amount of ¹²⁵I-Aβ₁₋₄₃bound to antibody was determined using a polyethylene glycol separationmethod. Results of the experiment are presented in Table 3.

The data in Table 3 demonstrate the ability of the purified 5A11monoclonal anti-Aβ antibody to bind a high percent of ¹²⁵I-Aβ₁₋₄₀. Thisbinding assay was used to screen clones and purified antibodies (Table3) for their ability to bind Aβ. Similar procedures can also serve asthe basis for a competitive displacement assay to measure the relativebinding strength of different unlabeled Aβ peptides. (Note: with veryefficient catalytic antibodies this binding assay may have to beperformed on ice to ensure that no cleavage of Aβ occurs during the 1 hincubation time.) The assay rapidly identified clones producing highaffinity anti-Aβ antibodies.

Monoclonal antibodies from hybridomas obtained using the phenylalaninestatine transition state Aβ-KLH antigen were screened by ELISA to assesstheir binding to both the normal Aβ₁₋₄₃ peptide and to the phenylalaninestatine transition state Aβ peptide. Two major patterns were found (FIG.13).

One group of antibodies (the left portion of FIG. 13) bound to theimmunizing transition state peptide and cross-reacted strongly with thenative Aβ₁₋₄₃ peptide (each was adsorbed directly onto the ELISA plate).The second group (the right portion) showed a high binding preferencefor the phenylalanine statine transition state Aβ peptide and reactedminimally with native Aβ₁₋₄₃.

Strong color reactions were obtained in this ELISA using only 10 μl ofhybridoma supernatant while Hy media alone or PBS gave a low background(FIG. 13). These results demonstrate that the comparative ELISA screen,although only a semi-quantitative measure of binding, provides a meansfor identifying monoclonal antibodies that are highly selective for, andmost reactive with, the transition state. Importantly, the experimentwas performed with carrier-free Aβ peptides adsorbed directly ontomicrotitre plates, indicating antibody specificity for Aβ peptide ratherthan carrier.

These findings indicate that several of the generated anti-Aβ transitionstate antibodies were unique. They bound to both the phenylalaninestatine- and normal-Aβ peptides. Their selective recognition of thetransition state and weaker cross-reaction with native Aβ₁₋₄₃ howeverindicates that this binding interaction is very different from thatshown by conventional anti-native Aβ antibodies. It further indicatesthat these new antibodies may be able to force the native AM peptideinto a conformation resembling the transition state for hydrolyticcleavage. Importantly, some of the antibodies which showed only minimalbinding to Aβ₁₋₄₃ in this ELISA, did display cross-reactivity with thenatural peptide using a highly sensitive ¹²⁵I-Aβ₁₋₄₃ binding assay(Table 3).

ELISAs were also performed to investigate the binding of anti-statineanalog antibodies to both the normal Aβ₁₋₄₃ peptide and to the statinetransition state Aβ peptide (FIG. 14). The antibodies bound to theC-terminal locus on these carrier-free Aβ peptides (adsorbed directly tothe microtitre plate) confirming their anti-peptide specificity. Most ofthe antibodies preferentially recognized the statine Aβ transitionstate, but cross-reacted with native Aβ₁₋₄₃. This indicates that thesenew antibodies are able to force the native Aβ peptide into aconformation resembling the transition state for hydrolytic cleavage ofits C-terminal amino acids. Such cleavage is predicted to convert Aβ₁₋₄₃into potentially less harmful shorter peptides, like Aβ₁₋₄₀ or Aβ₁₋₃₉.

Clone 11E9 had the strongest preference for the statine analog and maybe the most likely to have catalytic activity (FIG. 14). Several clonesdisplayed no difference in their reactivity with the native versusstatine transition state Aβ peptide. The clones were also tested withAβ₁₋₄₀ to identify antibodies which do not react with this shortened, 40amino acid version of Aβ (FIG. 14). Used therapeutically, suchantibodies should preferentially bind/cleave the less abundant, but morenoxious Aβ₁₋₄₃ species in the blood as opposed to the smaller and lessdetrimental Aβ₁₋₄₀.

Solid Phase and TLC Aβ Proteolytic Assays

A solid phase ¹²⁵I-labeled Aβ assay was developed to screenanti-transition state antibody hybridoma supernatants for specificproteolytic activity. The peptideCys-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Tyr-amide (SEQ IDNO: 5) which encompasses amino acids 14-25 of Aβ was radiolabeled andcoupled to a thiol-reactive, iodoacetyl-Sepharose gel to form anirreversible linkage. The product was incubated with anti-transitionstate antibody and assayed for the progressive release of soluble¹²⁵I-peptide from the solid phase matrix. Release of radioactivity fromthe ¹²⁵I-Aβ-Sepharose was used to identify catalytic activity (FIG. 15).The assay was verified by the ability of several different proteases torapidly hydrolyze this Sepharose-linked Aβ substrate. The peptide wasreadily accessible to proteolytic cleavage as revealed by a release ofsoluble ¹²⁵I-peptide that increased with incubation time.

The results presented in FIG. 15 indicate that the antibody-containingmedia of several clones released ¹²⁵I-peptide at a greater rate thanother clones from this fusion or the PBS and Hy medium controls. Largeamounts of these antibodies can be obtained, purified and tested athigher concentrations to achieve much faster rates of cleavage and toverify that the antibodies are acting in a catalytic mode usingconventional enzyme kinetics. By changing the composition of the¹²⁵I-peptide this same strategy can be used to assay antibodies reactivewith different regions of Aβ.

A thin layer chromatography-based autoradiography assay was devised toobtain more definitive evidence for antibody-mediated cleavage of Aβ.Selected anti-phenylalanine statine Aβ transition state clones wereexpanded and ascites production induced. The different monoclonalantibodies were isolated using protein A-Sepharose. Two ¹²⁵I-labeledpeptides, Aβ₁₋₄₀ and a 17-mer, encompassing amino acids 9-25 of Aβ, wereused to test for peptide cleavage. The antibodies were added to the¹²⁵I-peptides, allowed to incubate and the reaction mix spotted ontopolyamide thin layer sheets which were then developed in differentsolvents. The migration of ¹²⁵I-products was followed by exposing thesheet using a quantitative phosphoimager system Quantitation of thedifferent labeled peptide fragments produced indicated that addition ofthe antibodies to the Aβ peptides lead to significant break down of theAβ peptides compared to the untreated peptides (PBS).

Disaggregation of β-amyloid by Monoclonal Antibodies

The self-aggregation of synthetic Aβ peptides has been shown previouslyto lead to microscopic structures resembling amyloid plaques in thebrain (Solomon et al., Proc. Natl. Acad. Sci. USA 94: 4109-12 (1997);Solomon et al., Proc. Natl. Acad. Sci. USA 93: 452-5 (1996)) whichexhibit the same bright green fluorescence upon exposure to thioflavinT. These aggregates are very stable and usually require harsh detergentsor strong acids to dissolve. However, it has been demonstrated that thebinding of certain anti-Aβ monoclonal antibodies can effectively inhibitthe initial aggregation of this peptide and also disaggregate preformedAβ complexes (Solomon et al., Proc. Natl. Acad. Sci. USA 94: 4109-12(1997); Solomon et al., Proc. Natl. Acad. Sci. USA 93: 452-5 (1996)).

A radioactive assay was used to quickly screen the different monoclonalantibodies produced by the present experiments for an ability todissolve preformed Aβ aggregates, made with ¹²⁵I-labeled and unlabeledsoluble Aβ peptide. An aliquot of the labeled aggregate was incubatedwith either PBS, the 5A11 anti-Aβ antibody or an equal amount of anirrelevant mouse antibody (7D3, anti-human transferrin receptor), andthe level of released radioactivity was subsequently measured (Table 9).The Aβ-specific 5A11 antibody solubilized 80% of the Aβ aggregates whilean equal amount of the control antibody had only a minor effect,suggesting that the equilibrium was displaced by antibody-mediatedbinding of soluble Aβ. TABLE 9 Solubilization of ¹²⁵I-Aβ₁₋₄₀ Aggregateby Monoclonal Anti-Aβ Antibody ¹²⁵I-Aβ₁₋₄₀ in Ppt. Amount SolubilizedAddition (cpm) (% of PBS Control) PBS control 3,420 — +5A11 anti-Aβ 67680 +7D3 anti-TfR 2,458 27Production of Vectorized Anti-Aβ/Anti-Receptor Bispecific Antibodies

Anti-Aβ antibodies were linked to anti-transferrin receptor antibodies(anti-TfR) which served as vectors for delivery of the anti-Aβantibodies into the brain. The 7D3 mouse monoclonal antibody was used asthe anti-TfR part of the construct. 7D3 is specific for the humanreceptor and selectively immunostains cortical capillaries in normalhuman brain tissue (Recht et al., J. Neurosurg. 72: 941-945 (1990)).Antibody attachment to the receptor is not blocked by an excess of humantransferrin. The epitope recognized by this antibody is thereforedistant from the receptor-ligand binding site. Bispecific antibodiesconstructed with this 7D3 antibody and an anti-Aβ antibody are predictedto be useful for therapy in patients with Alzheimer's disease.

For studies in mouse models of Alzheimer's disease an anti-mousetransferrin receptor monoclonal antibody produced in the rat wasobtained. This antibody also appears to recognize a transferrin receptorepitope which does not involve ligand binding. The antibody thereforehas no effect on cell proliferation when using murine lines.

A series of functional assays were performed after completion of thesynthesis, purification and size analysis of theanti-Aβ/anti-transferrin receptor bispecific antibodies. The vectorizedbispecific antibody, composed of a rat monoclonal antibody directedagainst the mouse transferrin receptor plus the 5A11 mouse anti-Aβmonoclonal antibody, was tested for the ability to attach to transferrinreceptor bearing mouse cells. Both components of the bispecific antibodywere detected on the cell membrane by cytofluorimetry (FIG. 16) whenthis duplex was reacted with transferrin receptor positive mouse cellsand probed using either a rat IgG-specific or mouse IgG-specificfluorescent secondary antibody reagent.

The capacity of the hybrid reagent to bind ¹²⁵I -Aβ compared favorablywith that of the parent anti-Aβ antibody (Table 10). TABLE 10 ¹²⁵I-AβBinding to Bispecific Antibody ¹²⁵I-Aβ₁₋₄₀ Bound Addition (cpm) Control4,199 +anti-Aβ 23,301 +anti-Aβ/anti-receptor 22,850

To ensure that both of these binding activities resided on thebispecific antibody, transferrin receptor positive cells were treatedwith the hybrid reagent, unbound material was washed away, and then thecells with bound antibody was exposed to ¹²⁵I-Aβ₁₋₄₀. After washing awayunbound Aβ, the cell-bound radioactivity was compared to control cellswhich had been identically prepared except for omission of pretreatmentwith bispecific antibody. The results are presented in Table 11, andverify the dual specificity of this bispecific antibody by clearlyshowing that it can simultaneously attach to the cell membrane and bind.¹²⁵I-Aβ₁₋₄₀. TABLE 11 Bispecific Antibody-Mediated Binding of ¹²⁵I-Aβ toReceptor-Positive Cells Pretreatment of Cells ¹²⁵I-Aβ₁₋₄₀ Bound (cpm)None 2,367 +anti-Aβ/anti-transferrin receptor 11,476Transcytosis of Bispecific Antibody Into the Brain

A rat monoclonal anti-mouse transferrin receptor antibody was coupled toa mouse monoclonal antibody (obtained from American Type CultureCollection (ATCC TIB 219), also designated R17 217.1.3 (Cell. Immunol.83: 14-25 (1984)), so that the entry of this new vectorized bispecificconstruct into brain could be monitored. The bispecific antibody waslabeled with ¹²⁵I and injected i.v. into normal mice. After differentlengths of time the mice were sacrificed and the amount of¹²⁵I-bispecific antibody that crossed the blood-brain barrier andentered the brain was gauged by a mouse capillary depletion method(Friden et al., J. Pharm. Exper. Ther. 278: 1491-1498 (1996); Trigueroet al., J. Neurochem. 54: 1882-1888 (1990)).

The amount of vectorized bispecific antibody found in the brainparenchyma or brain capillary fractions was measured followingdifferential density centrifugation of the brain homogenate. Thesevalues were plotted as a function of time after i.v. injection (FIG.17). The time-dependent redistribution of radiolabeled bispecificantibody from the capillaries and into the parenchyma was consistentwith its passage across the cerebral endothelial blood-brain barrier(Joachim et al., Nature 341: 6239:226-30 (1989)). Even greateraccumulation in the parenchyma is expected to occur if the antibodiesattach to A8 in the cerebral plaques of plaque-bearing mice.

Monitoring the Brain Distribution of Bispecific Antibody in Live Mice

The ability to follow the entry and accumulation of vectorizedbispecific antibodies in the brain of live mice would greatly assist inthe development of the intracerebral treatment of plaque-bearing mice.Such a development would enable time-course studies and would greatlyreduce problems with inter-mouse variability. Preliminary studies with¹²⁵I-labeled bispecific antibodies were performed to determine ifimmunoscintigraphy was feasible in this system. As a first step, eitherthe radiolabeled vectorized bispecific antibody (¹²⁵I-R17/5A11) or anon-vectorized control bispecific antibody were administered to separatemice. Sequential brain images were accumulated at 1, 6, 24 and 48 hoursfollowing i.v. administration of the ¹²⁵I-labeled bispecific antibodyprobes. Although this technique suffered from a difficulty indetermining how much of the signal was due to the levels of blood-borneradioactivity circulating through the brain, significant distinctionswere noted in the brain of mice treated with the mouse transferrinreceptor reactive bispecific antibody versus those receiving the controlbispecific antibody. When the vectorized agent was used, brain levelsincreased between 1 and 6 hrs and then declined to a much lower level at24 and 48 hrs. Mice treated with the control displayed no increasebetween 1 and 6 hrs. The reason for decreased brain levels at 24 hrs andbeyond is not known but might be due to dehalogenation of the bispecificantibody probes so that free ¹²⁵I is released. Alternative methodsutilizing radioactive labels such as ¹¹¹In (Sheldon et al., Nucl. Med.Biol. 18: 519-526 (1991)) or ^(99m)Tc (Texic et al., Nucl. Med. Biol.22: 451-457 (1995)) attached to the vectorized bispecific antibody canbe utilized in future experiments if the use of iodine presents atechnical problem. This imaging technology will be useful fordetermining if smaller vectorized bispecific antibodies (eg. F(ab′)₂)with different physical properties and an altered biodistribution willpenetrate into the brain more effectively.

F(ab′)₂Heterodimers for Vector-Mediated Transport Into the Brain

The introduction of whole antibodies into the brain might be detrimentalif they were to fix complement and promote complement-mediated lysis ofneuronal cells. The development of smaller vectorized F(ab′)₂ bispecificreagents is expected to avoid this problem. It has been shown thataggregated Aβ itself can fix complement in the absence of any antibodyand that the resulting inflammation may contribute to the pathology ofAlzheimer's disease. The possibility of intracerebral antibody having asimilar effect would be greatly reduced by eliminating the Fc region ofthe antibody. Moreover, since coupling of Fab′ halves uses the intrinsichinge region cysteines, no extraneous substituent linkage groups need beadded.

Faster or more efficient entry into the brain represents anotherpotential advantage that smaller F(ab′)₂ or Fv₂ reagents provide forintracerebral delivery. Such modified bispecific agents can be preparedand compared to full-sized hybrid antibodies for their relativeeffectiveness in reaching the brain, crossing the blood-brain barrier,and affecting Aβ plaque development, by the methods described herein. Itis important to note, however, that only minor differences were foundwhen the capacity of differently-sized anti-transferrin receptorbispecific reagents for delivering toxins into cells byreceptor-mediated endocytosis was compared (Raso et al., J. Biol. Chem.272: 27623-27628 (1997)). This observation might indicate that littlevariation will be seen for transcytosis across the brain capillaryendothelial cells which form the blood-brain barrier. At the very leasthowever one would expect the two types of vectorized molecules to havedifferent biodistribution and plasma half-life characteristics(Spiegelberg et al., J. Exp. Med. 121: 323 (1965)).

Methods of the Invention

Antigen Synthesis. The statine and phenylalanine statine transitionstate peptides were synthesized using automated Fmoc chemistry.Fmoc-statine (Sta), [N-Fmoc-(3S,4S)-4-amino-3-hydroxy-6-methyl heptanoicacid] and Fmoc-“phenylalanine statine” (PhSta),[N-Fmoc-(3S,4S)-4-amino-3-hydroxy-5-phenylpentanoic acid] were purchasedcommercially. Each peptide was tested for purity by HPLC and itscomposition was verified by mass spectral and amino acid analysis.

The design strategy and methods for synthesizing phosphonamidate- andphosphonate-based transition state peptides are straightforward(Bartlett et al., Am. Chem. Society 22: 4618-4624 (1983); Bartlett etal., Biochemistry. 26: 8553-8561 (1987)). The N-terminal portion of thepeptide (N-acetyl-Cys-Met-Val-Gly) was made using standard automatedFmoc chemistry. After cleavage from the resin the N-acetyl tetrapeptidewas treated with pyridine disulfide to protect its sulfhydryl group. Anacid chloride of Cbz-glycine phosphonate monomethyl ester (Bartlett etal., Am. Chem. Society 22: 4618-4624 (1983); Bartlett et al.,Biochemistry 26: 8553-8561 (1987)) was coupled withVal-Val-Ile-Ala-amide which was synthesized by automated Fmoc chemistry.The last amino acid of Aβ, Thr, was omitted due to potential problemswith its unprotected hydroxyl group. The product, Cbz-Gly-PO₂⁻-NH-Val-Val-Ile-Ala-amide has a phosphonamidate (methyl ester) bondbetween the Gly and Val residues. Next, the Cbz blocking group wasremoved using hydrogen so that the protected N-acetyl-Cys-Met-Val-Glypeptide could be added to the amino terminal end of this transitionstate peptide by HBTU-activated peptide linkage. Treatment withmercaptoethanol and rabbit liver esterase was used to deblock thepeptide. Each key component in the synthetic scheme was tested forpurity by HPLC and its composition was verified by mass spectral andamino acid analysis.

A reduced peptide bond linkage was placed at the indicated sites in theAβ molecule. Automated Fmoc chemistry was used to begin synthesis of thepeptide. A pre-synthesized Fmoc amino aldehyde was then added manuallyand after the imide was reduced, automated synthesis was resumed (Meyeret al., J. Med. Chem. 38: 3462-3468 (1995)).

Coupling of Antigen to Carrier. The native and transition state Aβpeptides were coupled to maleimide-activated KLH by standard procedures(Partis et al., J. Pro. Chem. 2: 263-277 (1983)), in order to elicit animmune response. A Cys residue was strategically placed at the N— orC-terminal end of the peptides to provide a suitable linkage group forcoupling-them via a thioether bond to maleimide activated carrierproteins. This stable linkage attaches the peptide in a definedorientation. Addition of ˜20 peptides/KLH has been obtained based uponthe transition state amino acid content as determined by amino acidanalysis of the hydrolyzed conjugates (Tsao et al., Anal. Biochem. 197:137-142 (1991)).

Immunization of Mice. Standard protocols were used to immunize mice withthe KLH-linked Aβ peptides described in the preceding sections. Brieflythis procedure used i.p. injection of the different antigens emulsifiedin complete Freunds adjuvant, followed by a second course in incompleteFreunds adjuvant. Three days prior to the hybridoma fusion, the BALB/cmice were boosted i.v. with antigen in PBS. After ˜1 month animals weregiven a boost i.p. using the antigen emulsified with incompleteadjuvant. Serum from these animals was analyzed for anti-peptideantibodies by ELISA. BALB/c mice showing abundant antibody productionwere boosted by an i.v. injection with antigen and three days later theywere used to generate hybridoma clones that secrete monoclonalantibodies.

None of the mice immunized with Aβ vaccines or the anti-Aβascites-producing mice displayed ill effects even though some of thoseinduced antibodies cross-react with mouse Aβ and mouse amyloid precursorprotein.

Hybridoma Production I. A hybridoma fusion was performed using thespleen of a mouse immunized with the phenylalanine statine transitionstate Aβ-KLH antigen. Spleen cells from mice with the highest titre werefused with mouse myeloma NS-1 cells to establish hybridomas according tostandard procedures (Kohler et al., Nature 256: 495 (1975); R. H.Kennett, Fusion Protocols. Monoclonal Antibodies, eds. R. H. Kennett, T.J. McKearn and K. B. Bechtol. Plenum Press, New York. 365-367 pp.(1980)).

¹²⁵I-Aβ Binding Assay. Aβ₁₋₄₀and Aβ₁₋₄₃ were radiolabeled with ¹²⁵I andthe iodinated peptide then separated from unlabeled material by HPLC togive quantitative specific activity (˜2000 Ci/mmol) (Maggio et al.,Proc. Natl. Acad. Sci. 89: 5462-5466 (1992)). This probe was incubatedfor 1 h at 23° C. with either purified anti-Aβ antibodies or media takenfrom hybridoma clones producing anti-Aβ antibodies. A polyethyleneglycol separation method was used to detect the amount of ¹²⁵I-Aβ₁₋₄₃bound to antibody. By using serial dilution, this assay can providerelative binding affinities for the different hybridoma supernatants orpurified antibodies.

Solid Phase Aβ Proteolytic Assay. A solid phase ¹²⁵I-labeled ;o assaywas developed to screen anti-transition state antibody hybridomasupernatants for specific proteolytic activity. TheCys-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Tyr-amide peptide(SEQ ID NO: 5) encompassing amino acids 14-25 of Aβ was radiolabeledwith ¹²⁵I and the iodinated peptide was then separated from unlabeledmaterial by HPLC. The highly radioactive Aβ peptide was coupled to athiol-reactive, iodoacetyl-Sepharose gel to form an irreversiblelinkage. Antibodies were added to the labeled Aβ, which was then assayedfor progressive release of soluble ¹²⁵I-peptide from the solid phasematrix at pH 7, 25° C. This assay was verified by the ability of severaldifferent proteases in to rapidly hydrolyze this Sepharose-linked Aβsubstrate. Release of soluble ¹²⁵I-peptide increased with incubationtime.

Although Aβ is cleaved by several naturally occurring proteases,preliminary tests indicated that interference from high levels ofbackground hydrolysis was not a problem when assaying hybridomasupernatants of clones that did produce catalytic antibodies. A furtherprecaution that can be taken against exogenous proteases is carrying outall hybridoma cell fusions and cell culturing in serum-free media.

TLC Aβ Proteolytic Assay. A thin layer chromatography-basedautoradiography assay was used to obtain more definitive evidence forantibody-mediated cleavage of Aβ. Selected anti-phenylalanine statine Aβtransition state clones were expanded and ascites production induced.The different monoclonal antibodies were isolated using proteinA-Sepharose. The cleavage assay used ¹²⁵I-Aβ₁₋₄₀ and an ¹²⁵I-labeled17-mer, encompassing amino acids 9-25 of Aβ. Binding of the two¹²⁵I-labeled peptides to the purified monoclonal antibodies 5A11 and 6E2was examined using either a PEG precipitation assay or by aco-electrophoresis method. Peptide cleavage was tested by adding theantibodies to the ¹²⁵I-peptides, incubating and then spotting thereaction mix onto polyamide thin layer sheets. The chromatographs weredeveloped in different solvents (eg. 0.5 N HCl, 0.5 N NaOH or pH 7phosphate buffer) and the migration of ¹²⁵I-products was followed byexposing the sheet using a quantitative phosphoimager system.

Screen and Isolate Select Anti-Aβ Antibodies. An ELISA was used toinitially screen for anti-Aβ and anti-transition state Aβ peptidemonoclonal antibodies. Both the transition state peptide and thecorresponding natural Aβ peptide were adsorbed onto separate microtitreplates. The hybridoma supernatants were screened using two assays sothat the relative binding to both native and transition state Aβpeptides could be quantitated. Clones producing monoclonal antibodiesthat preferentially recognized the transition state or bound Aβ withhigh affinity were selected for expansion and further study.

Propagation and Purification of Monoclonal Antibodies. Selected clonesproducing anti-Aβ antibodies and clones producing anti-receptorantibodies were injected into separate pristane-primed mice. Asciteswere collected and the specific monoclonal antibodies isolated.Purification of antibodies from ascites was accomplished using a ProteinA column or alternatively, antibodies were isolated from ascites fluidby (NH₄)₂SO₄ precipitation and passage over an S-300 column to obtainthe 150 kDa immunoglobulin fraction. Monovalent Fab fragments wereprepared and isolated by established methods. Their purity was evaluatedby SDS-PAGE under reducing and non-reducing conditions. 50-100 mg ofpurified monoclonal antibody was routinely obtained from eachascites-bearing mouse.

Further Characterization of Catalytic Activity on Aβ Substrates. Tofully define the hydrolytic properties of the isolated anti-transitionstate antibodies some very important controls can be run. First theability to completely block catalytic antibody activity with theappropriate transition state peptide can be verified. This non-cleavable“inhibitor” should bind much more tightly to the antibody combiningsites and thereby prevent substrate binding or cleavage. Substratespecificity can be further established by showing no cleavage of a shamAβ peptide having a different amino acid sequence. The products ofhydrolysis can also be fully characterized by HPLC, amino acid and massspectral analysis. Control antibodies that are not directed against thetransition state Aβ can be tested and confirmed to produce no catalysis.Finally, catalytic activity can be shown to reside in the purified Fabfragments of the anti-transition state antibody.

Purified Anti-Aβ Antibodies Dissolve Preformed Aβ Aggregates. (Walker etal., Soc. Neurosci. Abstr. 21: 257 (1995), Zlokovic, B. V., LifeSciences 59: 1483-1497 (1996)). Aβ precipitates were formed and measuredin vitro (Yankner et al., Science 250: 279-282 (1990), Kowall et al.,Proc. Natl. Acad. Sci. 88: 7247-7251 (1991)). A radioactive assay wasused to quickly screen the different monoclonal antibodies produced foran ability to dissolve preformed Aβ aggregates. After adding-¹²⁵I-Aβ tounlabeled soluble peptide, aggregates were formed by bringing thesolution to pH 5 or by stirring it overnight in PBS. An aliquot of thelabeled aggregate was incubated for 1 hr with either PBS, the 5A11anti-Aβ antibody or an equal amount of an irrelevant mouse antibody(7D3, anti-human transferrin receptor). After centrifugation, the levelof radioactivity in the precipitate was measured.

Generation of Vectorized Anti-Aβ/Anti-Receptor Bispecific Antibodies.The anti-Aβ antibodies were chemically coupled to anti-human-transferrinreceptor and anti-mouse transferrin receptor antibodies by differentmethods (Raso et al., J. Biol. Chem. 272: 27623-27628 (1997); Raso etal., Monoclonal antibodies as cell targeted carriers of covalently andnon-covalently attached toxins. In Receptor mediated targeting of drugs,vol. 82. G. Gregoriadis, G. Post, J. Senior and A. Trouet, editors. NATOAdvanced Studies Inst., New York. 119-138 (1984)). A rapid thioetherlinkage technique was used to form strictly bispecific hybrids usingTraut's reagent and the heterobifunctional SMBP reagent. One componentwas sparingly substituted with thiol groups (SH). These readily reactedto form a thioether linkage upon mixture with the maleimido-substituted(M) second component following the reaction:

-   -   Ab_(A)-SH+Ab_(B)-M→Ab_(A)-S-Ab_(B)

Gel filtration of the reaction mixture on an S-300 column yielded thepurified dimer which was 300 kDa and had two sites for binding Aβ plustwo sites for attachment to transferrin receptors on brain capillaryendothelial cells. Non-targeted control hybrids were formed by linking anonspecific MOPC antibody to the anti-Aβ antibody. This hybrid antibodydoes bind Aβ, but, being non-reactive with transferrin receptors, shouldnot cross the blood-brain barrier.

F(ab′)₂ fragments of the two different antibody types can similarly bethioether-linked to form Fc-devoid reagents that cannot bind complementwhich might otherwise cause neurotoxic effects. These smaller bispecifichybrids (100 kDa) can be formed by reducing the intrinsic disulfideswhich link the heavy chains of F(ab′)₂ fragments (Raso et al., J.Immunol. 125: 2610-2616 (1980)). The thiols generated are stabilized andEllman's reagent (E) is used to activate these groups on one of thecomponents (Brennan et al., Science 229: 81-83 (1985)). Exclusivelybispecific F(ab′)₂ hybrids can be formed upon mixing the reduced Fab′with an activated Fab′ having the alternate specificity according to thereaction:

-   -   Fab′_(A)-SH+Fab′_(B)-SS-E→Fab′_(A)-SS-Fab′_(B)+E-SH

Purification on an S-200 column will isolate hybrids with one site forbinding Ad and one site for interaction with the target epitope on thebrain capillary endothelial cells.

A similar approach can be used to make even smaller disulfide-linkedsingle chain Fv heterobispecific dimers, Fv_(A)-SS-FV_(B) (50 kDa), tocross the blood-brain barrier. Soluble Fvs can be constructed to possessa carboxyl-terminal cysteine to facilitate the disulfide exchange shownin the reaction below, and create 50 kDa heterodimers exclusively:

-   -   Fv_(A)-SH+Fv_(B)-SS-E→Fv_(A)-SS-Fv_(B)+E-SH

In side by side comparisons between whole antibody and either Fab′ or Fvbased bispecific reagents, the latter have proven to be moderately moreeffective on a molar basis for cell uptake via the transferrinreceptor-mediated pathway (Raso et al., J. Biol. Chem. 272: 27623-27628(1997)). Since these smaller constructs are monovalent for thecell-surface epitope, those findings dispel the notion thatcross-linking of two surface receptors is necessary for the cellularuptake of immunocomplexes.

Functional Assays for Dual Binding Activity of Bispecific Antibodies.The capacity of the hybrid reagent to bind ¹²⁵I-Aβ was compared withthat of the parent anti-Aβ antibody in a standard PEG binding assay (seeTable 10 for binding assays).

The ability of the appropriate bispecific antibodies to attach totransferrin receptor bearing human or mouse cells was confirmed bycytofluorimetry. The bispecific antibody was reacted with transferrinreceptor positive human or mouse cells and probed using either a ratIgG-specific or mouse IgG-specific fluorescent secondary antibodyreagent.

Measurement of Aβ Binding Using ¹²⁵I-Aβ and a Polyethylene GlycolSeparation. To ensure bispecificity, hybrid reagents were tested for acapacity to mediate the attachment of ¹²⁵I-Aβ to receptor-bearing cells.Transferrin receptor positive cells were treated with the hybridreagent, washed away unbound material and then exposed these cells to¹²⁵I-Aβ₁₋₄₀. The cells were washed and the amount of cell-boundradioactivity was compared to control cells which had been identicallyprepared except that pretreatment with bispecific antibody was omitted.

Capillary Depletion. The bispecific antibody was labeled with ¹²⁵I andinjected i.v. into normal mice. After different lengths of time the micewere sacrificed and the amount of ¹²⁵I-bispecific antibody that crossedthe blood-brain barrier and entered the brain was gauged by a mousecapillary depletion method (Friden et al., J. Pharm. Exper. Ther. 278:1491-1498 (1996); Triguero et al., J. Neurochem. 54: 1882-1888 (1990)).The amount of vectorized bispecific antibody found in the brainparenchyma or brain capillary fractions was measured followingdifferential density centrifugation of the brain homogenate. Thesevalues were plotted as a function of time after i.v. injection.Progressive passage from capillaries into the parenchyma indicatesactive transcytosis across the blood-brain barrier.

Immunoscintigraphy. A non-invasive method for monitoring intracerebraldelivery process which involves visualizing the entry of a radiolabeledbispecific antibody into the brain of live mice, can also be used.Radiolabeled vectorized bispecific antibody (¹²⁵I-R17/5A11) or anon-vectorized control bispecific antibody were administered to separatemice. Sequential brain images were accumulated at 1, 6, 24 and 48 hoursfollowing i.v. administration of the ¹²⁵I-labeled bispecific antibodyprobes. The animals were chemically immobilized during exposure usingketamine/xylazine anesthesia. This imaging technology could be veryuseful for determining if circulating anti-Aβ antibodies will preventi.v. administered ¹²⁵I-Aβ from entering the brain. Digital scintigraphydata was quantified using standards and the integration functionsprovided in the analysis software.

1. A method for inhibiting the formation of,8-amyloid plaques in thebrain of a human, the method comprising: a) providing a β-amyloidepitope; and b) administering the epitope of step a) to the human underconditions appropriate for the stimulation of an immune responsedirected toward the epitope, the immune response being characterized bythe generation of circulating antibodies which bind specifically to theepitope present on endogenous, β-amyloid in the human.
 2. The method ofclaim 1 wherein the epitope of step a) is administered in an adjuvantformulation.
 3. The method of claim 2 wherein the adjuvant formulationcomprises oil emulsion.
 4. The method of claim 1 wherein the binding ofcirculating antibodies to endogenous β-amyloid detectably alters theequilibrium distribution of free β-amyloid in circulation versus freeβ-amyloid in the brain of the human.
 5. The method of claim 1 whereinthe epitope of β-amyloid is linked to an immunogenic carrier moiety. 6.The method of claim 5 wherein the immunogenic carrier moiety is KeyholeLimpet Hemocyanin (KLH).
 7. The method of claim 1 wherein the epitope isprovided as β-amyloid peptide Aβ₁₋₄₃.
 8. The method of claim 1 whereinthe epitope is provided as β-amyloid peptide Aβ₁₋₄₀.
 9. The method ofclaim 1 wherein the epitope is provided as a peptide fragment ofβ-amyloid, the peptide fragment being derived from the N-terminal regionof the β-amyloid peptide Aβ₁₋₄₃.
 10. The method of claim 1 wherein theepitope is provided as a peptide fragment of β-amyloid, the peptidefragment being derived from the central region of the β-amyloid peptideAβ₁₋₄₃.
 11. The method of claim 1 wherein the epitope is provided as apeptide fragment of β-amyloid, the peptide fragment being derived fromthe C-terminal region of β-amyloid peptide.
 12. A method for inhibitingthe formation of β-amyloid aggregates and plaques in the brain of ahuman, the method comprising: a) providing a plurality of peptidefragments derived from β-amyloid peptide Aβ₁₋₄₃, each peptide fragmentcomprising one or more β-amyloid epitopes; and b) administering theplurality of peptide fragments of step a) to the human under conditionsappropriate for the stimulation of an immune response directed towardthe β-amyloid epitopes, the immune response being characterized by thegeneration of circulating antibodies which bind specifically to one ormore epitopes present on endogenous, β-amyloid in the human.
 13. Themethod of claim 12 wherein at least one epitope is provided as a peptidefragment of β-amyloid, the peptide fragment being derived from theN-terminal region of the β-amyloid peptide Aβ₁₋₄₃.
 14. The method ofclaim 12 wherein at least one epitope is provided as a peptide fragmentof β-amyloid, the peptide fragment being derived from the central regionof the β-amyloid peptide Aβ₁₋₄₃.
 15. The method of claim 12 wherein atleast one epitope is provided as a peptide fragment of β-amyloid, thepeptide fragment being derived from the C-terminal region of β-amyloidpeptide.